Redox signaling plays an important role in the positive regulation of angiogenesis by vascular endothelial growth factor, but its role in signal transduction by angiogenesis inhibitors is less clear. Using muscle explants in 3D culture, we found that explants from mice lacking TSP1 exhibit exaggerated angiogenic responses to an exogenous NO donor, which could be reversed by providing exogenous TSP1. To define the basis for inhibition by TSP1, we examined the effects of TSP1 on several pro-angiogenic responses of endothelial and vascular smooth muscle cells to NO. NO has a biphasic effect on proliferation of both vascular cell types. The positive effect at low doses of NO is sensitive to inhibition of cGMP signaling and to picomolar concentrations of TSP1. NO stimulates both directed (chemotactic) and random (chemokinetic) motility in a cGMP-dependent manner. TSP1 potently inhibits chemotaxis stimulated by NO. Low doses of NO also stimulate adhesion of vascular cells on type I collagen in a cGMP-dependent manner. TSP1 potently inhibits this response both upstream and downstream of cGMP. NO-stimulated vascular cell responses are inhibited by recombinant type 1 repeats of TSP1 and by a CD36 agonist antibody but not by the N-terminal portion of TSP1, suggesting that CD36 or a related receptor mediates these effects. These results demonstrate a potent antagonism between TSP1 and pro-angiogenic signaling downstream of NO. Further elucidation of this inhibitory signaling pathway may identify new molecular targets to regulate pathological angiogenesis. Although ligation of CD36 by antibodies, recombinant type 1 repeats of TSP1, or CD36-binding peptides was sufficient to inhibit nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells, picomolar concentrations of native TSP1 similarly inhibited NO signaling in vascular cells from wild type and CD36 null mice. Ligation of the TSP1 receptor CD47 by recombinant C-terminal regions of TSP1, TSP1 peptides, or CD47 antibodies was also sufficient to inhibit NO-stimulated phenotypic responses and cGMP signaling in vascular cells. TSP1 did not inhibit NO signaling in CD47 null vascular cells or NO-stimulated vascular outgrowth from CD47 null muscle explants in 3D cultures. Furthermore, the CD36-binding domain of TSP1 and anti-angiogenic peptides derived from this domain failed to inhibit NO signaling in CD47 null cells. Therefore, ligation of either CD36 or CD47 is sufficient to inhibit NO-stimulated vascular cell responses and cGMP signaling, but only CD47 is necessary for this activity of TSP1 at physiological concentrations.